Ubiquitination is an important form of post-translational modification that can determine a protein's fate. While ubiquitin itself is a small and conserved protein, its covalent conjugation to protein substrates and to other ubiquitin molecules is a tightly controlled process involving complex cellular machinery. Perhaps the most prominent and well-known function of ubiquitin is to target a protein for degradation by the 26S proteasome. This is done via isopeptide bond formation between the carboxy-terminal Gly on the ubiquitin and ε-amino group of lysine side chains of the protein substrate.
The ubiquitin-substrate system is further diversified via the process of polyubiquitination, during which a ubiquitin molecule's C-terminal Gly is conjugated with one of the seven Lys residues on another ubiquitin (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, or Lys63) or with the N-terminus to form linear chains. While Lys48-linked and Lys11-linked polyubiquitination has been shown mostly to target protein substrates for 26S-mediated degradation, other functions of ubiquitination continue to unfold. For example, Lys63-polyubiquitination is involved in DNA repair and endocytosis, while both Lys63-linked and linear polyubiquitination has been demonstrated to regulate immunity via NF-κB activation. Accordingly, the biological complexity of ubiquitination is complex and suggests that enzymes involved in this process must have remarkable specificity to correctly carry out their unique functions. E1, E2, and E3 are enzymes that together facilitate the multistep process of ubiquitin-substrate conjugation, and deubiquitinat-ing enzymes (deubiquitinases or DUBs) carry out the reverse steps of breaking the isopeptide bond.
There are approximately 100 DUBs known in the human genome, any of which could be playing a key role in the ubiquitin-proteasome system as well as other biological processes. The main functions for which DUBs are responsible include: (a) liberation of ubiquitin from protein substrates (e.g., to remove degradation signal), (b) editing of polyubiquitin signal on protein substrates to change the fate of the protein, (c) disassembling polyubiquitin chains to free up ubiquitin monomers, (d) cleaving ubiquitin precursors or adducts to regenerate active ubiquitin.
The DUBs are subdivided into five families: ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific proteases (USPs), ovarian tumor proteases (OTUs), Josephins and JAB1/MPN/MOV34 metalloenzymes (JAMM/MPN+). The first four families (UCH, USP, OTU, and Josephin) are cysteine proteases, while JAMM/MPN+ are zinc metalloproteases. Among these, UCHs and USPs are the best characterized, and USPs represent more than half of the known human DUBs. The reaction mechanism of the cysteine protease DUB families is the same as that of the cysteine protease superfamily. Each enzyme active site requires the interplay between three conserved residues forming the catalytic triad: a cysteine, a histidine, and an aspartic acid. The initial attack creates a negatively charged transition state stabilized by the oxy-anion hole. The intermediate is a thiohemiacetal stabilized through interactions with the active site residues as an incoming water molecule liberates the lysine side chain from the conjugated ubiquitin or substrate. A nucleophilic attack of the water creates another negatively charged transition state, which rearranges to free up the carboxylate terminus of the N-terminal ubiquitin and restores the enzyme to its apo form.
Recently, increased biological understanding has led to numerous DUBs being implicated in various diseases spanning oncology, neurodegeneration, hematology, and infectious diseases. Most recently, Bingol et al. carried out elegant experiments in vitro and in vivo to illustrate the role of USP30 as an antagonist of Parkin-mediated mitophagy, suggesting the inhibition of USP30 as a potential therapy for Parkinson's disease (PD). The hunt for DUB antagonists is thus actively carried out by academic and pharmaceutical companies alike. This is illustrated through chemically diverse small molecules that have been reported to inhibit one or more of the UCH and USP family members.
Indeed, the covalent attachment of ubiquitin to proteins is an important step in the degradation of proteins via the 26S proteasome. See e.g., Metzger M B et. al. [J Cell Sci. 2012; 125:531-7] Deubiquitinating enzymes (DUBs), also known as deubiquitinating peptidases, deubiquitinating isopeptidases, deubiquitinases, ubiquitin proteases, ubiquitin hydrolases, ubiquitin isopeptidases, are a large group of proteases that cleave ubiquitin from proteins and other molecules. [Nijman S M, et al. Cell. 2005; 123:773-86] DUBs can prevent the degradation of proteins by cleaving the peptide or isopeptide bond between ubiquitin and its substrate protein. In humans there are nearly 100 DUB genes, which can be classified into two main classes: cysteine proteases and metalloproteases. The cysteine proteases comprise ubiquitin-specific proteases (USPs), ubiquitin C-terminal hydrolases (UCHs), Machado-Josephin domain proteases (MJDs) and ovarian tumour proteases (OTU). The metalloprotease group contains only the Jab1/Mov34/Mpr1 Pad1 N-terminal+ (MPN+) (JAMM) domain proteases.
Ubiquitin-specific-processing protease 7 (USP7), also known as ubiquitin carboxyl-terminal hydrolase 7 or herpesvirus-associated ubiquitin-specific protease (HAUSP), is an enzyme that in humans is encoded by the USP7 gene. USP7 or HAUSP is a DUB enzyme that cleaves ubiquitin from its substrates. Since ubiquitylation (polyubiquitination) is most commonly associated with the stability and degradation of cellular proteins, USP7 activity generally stabilizes its substrate proteins. [Shi D et. al. Cancer Biology and Therapy 2010 10:8 737-747]
USP7 is most popularly known as a direct antagonist of Mdm2, the E3 ubiquitin ligase for the tumor suppressor protein, p53. Normally, p53 levels are kept low in part due to Mdm2-mediated ubiquitylation and degradation of p53. In response to oncogenic insults, USP7 can deubiquitinate p53 and protect p53 from Mdm2-mediated degradation, indicating that it may possess a tumor suppressor function for the immediate stabilization of p53 in response to stress.
In addition, USP7 also plays a key role in the immunoregulatory transcription factor protein FOXP3. By de-ubiquitylating and preserving FOXP3, USP7 increases T regulatory cell (Treg) mediated suppression of tumor-infiltrating T effector cells, the latter being associated with improved clinical outcome for many solid tumors. Thus, USP7 functions to limit immune cell-mediated antitumor defenses. The observation that the accumulation of FOXP3+ Treg cells at the tumor or in draining lymph nodes signals poor prognosis further highlights the significance of this recently described second oncogenic mechanism of USP7. Thus, inhibitors of USP7 can exert in vivo antitumor activity by: 1) directly inhibiting tumor cell proliferation via Hdm2 and other targets; and 2) suppressing T regulatory cells via FOXP3, thereby facilitating the antitumor function of T effector cells.
Another important role of USP7 function involves the oncogenic stabilization of p53. Oncogenes such as Myc and E1A are thought to activate p53 through a p19 alternative reading frame (p19ARF, also called ARF)-dependent pathway, although some evidence suggests ARF is not essential in this process. A possibility is that USP7 provides an alternative pathway for safeguarding the cell against oncogenic insults.
USP7 can deubiquitinate histone H2B and this activity is associated with gene silencing in Drosophila. USP7 associates with a metabolic enzyme, GMP synthetase (GMPS) and this association stimulates USP7 deubiquitinase activity towards H2B. The USP7-GMPS complex is recruited to the polycomb (Pc) region in Drosophila and contributes to epigenetic silencing of homeotic genes.
USP7 was originally identified as a protein associated with the ICP0 protein of herpes simplex virus (HSV), hence the alternate name Herpesvirus Associated USP (HAUSP). ICP0 is an E3-ubiquitin ligase that is involved in ubiquitination and subsequent degradation of itself and certain cellular proteins. USP7 has been shown to regulate the auto-ubiquitination and degradation of ICP0.
More recently, an interaction between USP7 and the EBNA1 protein of Epstein-Barr virus (EBV) (another herpes virus) was also discovered. This interaction is particularly interesting given the oncogenic potential (potential to cause cancer) of EBV, which is associated with several human cancers. EBNA1 can compete with p53 for binding USP7. Stabilization by USP7 is important for the tumor suppressor function of p53. In cells, EBNA1 can sequester USP7 from p53 and thus attenuate stabilization of p53, rendering the cells predisposed to turning cancerous. Compromising the function of p53 by sequestering USP7 is one way EBNA1 can contribute to the oncogenic potential of EBV. Additionally, human USP7 was also shown to form a complex with GMPS and this complex is recruited to EBV genome sequences. USP7 was shown to be important for histone H2B deubiquitination in human cells and for deubiquitination of histone H2B incorporated in the EBV genome. Thus, USP7 may also be important for regulation of viral gene expression. The fact that viral proteins have evolved so as to target USP7, underscores the significance of USP7 in tumor suppression and other cellular processes.